Abstract

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.

Imaging orientation of fluorescent single molecules with instantaneous FluoPolScope. (A) Coordinate system used to parameterize single dipoles and their ensembles (thick green line through origin) by their net orientation in the focal plane (XY), net tilt relative to the optical axis of the microscope (Z), and wobble during the exposure. (B) Schematic of the microscope. AP, aperture; CL, collimating lens; DM, dichroic mirror; IF, interference filter; PBS, polarization beam splitter; TL, tube lens. (C) Polarization resolved fluorescence images of GFP molecules attached to coverslip. (D) Enlarged images of single GFP molecule identified by the outlined square in C. Right shows the computed dipole orientation as a magenta line overlaid on the sum of all polarization-resolved images. (E) Particle intensities (Top), background intensities (Middle), and orientation and polarization factor (Bottom) of the single GFP molecule shown in D that was recorded for more than 3 min. Inset in Middle identifies the pixels used to estimate particle intensities (area enclosed by the yellow line) and background intensity (circular area between two blue lines) in each quadrant. (F) Fluorescence images of λ phage DNA stained with 50 nM TOTO-1 (Left) and the image with orientations of polarized fluorescence of detected particles shown by magenta lines (Right). (Scale bars: magenta in C and F, polarization factor = 1; white in C and F, 1 μm.)

Position and orientation of AF488-phalloidin bound to F-actin in vitro. (A) Fluorescence image of F-actin stained with AF488-phalloidin (2 nM in actin polymerization buffer) at the start of image sequence. (B) The image as in A with orientations of polarized fluorescence shown by magenta lines. *Representative particle used to illustrate tracking of position, intensity, orientation, and polarization factor. #Measured orientation aligns with a straight stretch of the filament. (C) Positions of centroid of representative particle (* in B) over time connected by a black line. Hue of the dots represents the particle intensity in photoelectrons (pe; color map in Inset). Centroids are overlaid on a magnified, averaged fluorescence image from the start to the end of the track. Dashed white line shows ensemble orientation (ϕe) of polarized fluorescence of the pixels comprising the particle over the first three frames. The ensemble orientation is used to define the local filament orientation (ϕf). (D) Kymograph of fluorescence particles along the long axis of the white rectangle in A. Position of the particles in the filament is plotted in the vertical direction, and time is along the horizontal direction. The tracks of two particles are shown in magenta. *Representative particle identified in B and C. (E) Time-dependent intensity change of the representative particle. (F) Time-dependent orientation change of the representative particle relative to local filament orientation ϕf. **When the ensemble reduces to a single fluorophore of AF488, its orientation remains close to the filament orientation. (G) Polarization factor of the representative particle. **The polarization factor increases and becomes noisier when the ensemble reduces to single fluorophore. In E–G, the dotted lines show the intensity, orientation, and polarization factor after the track bleaches to the background, respectively. The uncertainty (±SD) in the measured quantities because of shot noise is shown by blue error bars at chosen time points. In some graphs, blue error bars are too small to be clearly visible. (H) Histogram of particle intensities over ∼2,600 individual observations (60 tracked particles). Peaks at multiples of 450 photoelectrons (arrowheads) suggest the intensity of a single AF488. (I) Scatterplot of polarization orientation relative to filament axis vs. intensity for the same observations. Individual scatter points are colored by their polarization factor p according to legend. Total number of scatter points corresponds to the same number of individual observations in H. (Scale bars: white in A and B, 1 μm; magenta in B, polarization factor = 1; C, 100 nm.)

Position and orientation of AF488-phalloidin bound to F-actin during retrograde flow in live cells. (A) Maximum intensity projection of F-actin network sparsely labeled with AF488-phalloidin at the leading edge of a migrating human keratinocyte (HaCaT cell). Maximum intensity projection over the 20-min-long movie shows complete coverage of the leading edge consisting of actin arcs and lamellipodium. (B) The direction (ω) and the speed (s) of the movement estimated from the motion of the tracked particle are shown by green arrows. The direction of the arrow indicates the local direction of the movement, and the length of the arrow indicates the speed. Trajectories of a few fluorescent particles are shown by white lines. (C) Orientation of polarized fluorescence (ϕ) and polarization factor (p) are shown by magenta lines. Orientation of the lines represents the polarization orientation, and the length of the lines represents the polarization factors. (B and C) Yellow circles, squares, and diamonds mark the starting positions of three tracks plotted in D, whereas the white lines indicate the trajectories of the particle up to the chosen time point of t = 4 min. (D) Time-resolved fluctuations in the fluorescence polarization orientation of individual particles tracked in B and C. The orientation is plotted relative to the net direction of the track computed by vector averaging of all flow vectors along the track. (E) Temporal projection of flow speed is shown as a scatterplot of dots placed at their detected positions and colored according to their local retrograde flow speed. The color is assigned according to the color scale in the upper left. (F) Temporal projection of moving directions is constructed as described in E but using the color wheel with periodicity of 360° shown in the upper left. (G) Temporal projection of orientation of polarized fluorescence constructed as described in E but using the color wheel with periodicity of 180° shown in the upper left. (F and G) The dashed white lines identify the region of interest used for statistical analysis in H. (E–G) Bands parallel to leading edge corresponding to actin arcs are marked as arcs, and the band corresponding to lamellipodium is marked as lam. (H) Normalized histogram of retrograde flow directions (green line) and polarization orientations (magenta line) of AF488-phalloidin particles in the areas enclosed by white dashed lines in F and G. The distribution of retrograde flow direction and polarization orientation is plotted relative to the mean retrograde flow direction (dashed green line) within the region of interest. (Scale bars: white in A–C and E–G, 1 μm; green in B, flow speed (s) = 25 nm/s; magenta in C, polarization factor = 1.)

Position and orientation of single septin-conGFP⊥ particles in live A. gossypii. Data are presented in the same way as in . (A) Fluorescence images of septin-conGFP⊥ expressed in A. gossypii. (B) The same fluorescence image in A with fluorescence polarization orientations indicated by magenta lines measured at the start of an image sequence. The ensemble orientation (ϕe) of polarized fluorescence of particles is roughly perpendicular to the visible long axis of slightly bent septin bars. The local orientation of septin bars (ϕf) is defined to be perpendicular to local ensemble orientation. (C) Positions of centroid of representative particle (* in A) over time. Color of particles indicates intensity in photoelectrons (pe). (D) Kymograph of fluorescence particles along the long axis of the white rectangle in A. Time-dependent changes of intensity, orientation, and polarization factor of representative particle are plotted over time in E–G, respectively. Blue error bars represent SDs caused by shot noise. **This particle exhibits change in polarization factor and orientation synchronous with drop to single-molecule intensity that cannot be explained by uncertainty caused by shot noise. (H) Histogram of particle intensities before they bleached to the background from 1,200 particle observations (52 tracks). The histogram suggests that the single septin-conGFP⊥’s intensity is ∼350 photoelectrons. (I) Scatterplot of orientation (relative to ϕf) vs. intensity (above background) of the same particles. (Scale bars: white in B, 1 μm; magenta in B, polarization factor = 1; green in C, 100 nm.)

Fluctuation analysis of orientation, position, and intensity of Cdc12-conGFP⊥, PH-GFP, and GFP particles. Cdc12-conGFP⊥s were imaged continuously with 500-ms exposure and 1.1-μW/μm2 laser power, PH-GFPs were imaged continuously with 100-ms exposure and 5.5-μW/μm2 laser power, and GFP on poly-d-lysine–coated coverslip was imaged at 5-s intervals with 500-ms exposure and 1.1-μW/μm2 laser power only during exposure. (A) Trajectories of representative particles. Nascent or newly bound Cdc12-conGFP⊥ to septin bars, Cdc12-conGFP⊥s that were preassembled in septin bars, nascent PH-GFP in Ashbya, and nascent in vitro GFP bound to the surface of glass coverslips in columns 1–4, respectively. For nascent Cdc12-conGFP⊥, PH-GFP, and in vitro GFP, trajectories are shown from the time that they arrive within the TIR depth to their disappearance. For preassembled Cdc12-conGFP⊥, the trajectory is shown back in time from the time of disappearance. The dashed lines in Cdc12-conGFP⊥ plots show the ensemble polarization orientation ϕe at the start of the acquisition; ϕe is computed by averaging particle intensities in the first three frames of the track. (B–D) Fluctuations in intensity, position, and orientation, respectively. Fluctuations were analyzed across the ensemble of n particle tracks aligned at the frame of appearance or the frame of disappearance: n = 48 for nascent Cdc12-conGFP⊥, n = 52 for preassembled Cdc12-conGFP⊥, n = 58 for PH-GFP, and n = 95 for GFP. Unlike and , the fluctuations are analyzed relative to the ensemble orientation (ϕe), because filament orientation (ϕf) is not applicable to PH-GFP and immobile GFP measurements. (B) Mean (solid line) and SD (shaded area around mean) of particle intensity (blue) and background (bg) intensity (red) among the aligned tracks. (C) Mean and SD of squared displacement relative the position at t = 0. For Cdc12-conGFP⊥, the vector squared displacement is partitioned into components parallel (||ϕe) and perpendicular (⊥ϕe) to the ensemble orientation. (D) Mean and fluctuations of polarization orientation across the aligned tracks computed using circular statistics as described in . Orientation within each track is relative to its ensemble orientation as described in A. The shot noise-induced uncertainty in mean orientation is shown by the dotted line.